This proposal;l is concerned with delineation of the remaining structural and mechanistic questions on pyruvate decarboxylase (PDC, E.C.4.1.1.1), perhaps the simplest nonoxidative decarboxylase requiring thiamin diphosphate (ThDP, the vitamin B1 coenzyme, of fundamental significance in human metabolism). The brewers'yeast enzyme was purified as fully active alpha4 and beta4 homotetramers and the alpha4 structure was crystallized by the AI and collaborators at the VA Hospital in Pittsburgh. A 2.4 electron density map of this enzyme is more than 95% complete, including the coenzyme binding site. With this map already in hand, for the first time on this representative of a large group of alpha-keto acid decarboxylases, rational experiments can be designed to probe: a. the conformation of the enzyme-bound coenzyme in the absence of substrate, as well as in some of the three putative covalent substrate-coenzyme complexes; b. the activation towards catalysis (in the absence and presence of substrate activation) of the two aromatic rings (thiazolium and 4-aminopyramidine), and test of the hypothesis that both rings (not only the thiazolium) are participants in catalysis; c. the environment of the coenzyme and the function of amino acids surrounding it in catalysis, for example determining if surrounding hydrophobic amino acids provide a "solvent effect" thereby lowering the transition state energies of a number of key steps, as suggested by model studies ; and d. structural bases and structural as well as mechanistic consequences of substrate activation (using among other tools pyruvamide, a nondecarboxylatable substrate surrogate that is capable of shifting the enzyme from a low to a high activity form). Most prominent among the tools to address these questions will be (all of these are "in hand" as of writing): a. x-ray crystallographic methods (this part of the research is a collaborative effort with Furey, Sax and coworkers at the VA Hospital Biocrystallography Lab/Univ. of Pittsburgh); b. site-directed mutagenesis of amino acids found near the catalytic and regulatory sites based on the x-ray crystallographic and mechanistic information; c. further elucidation of the chemistry and enzyme-bound environment of the enamine, one of the three ThDP-substrate covalent complexes on PDC; 4. modeling based on the x-ray coordinates to help refine mechanistic models, to help design even more insightful experiments, and to compare the three dimensional models of two enzymes with very high sequence homology to PDC's (pyruvate oxidase and acetolactate synthetase) that have identical mechanisms through pyruvate decarboxylation, but diverge significantly thereafter. Undoubtedly, the fundamental questions to be resolved by the proposed research will be of profound interest and significance to many other research groups working on thiamin around the globe.